ABSTRACT The interactions between previous life stress and sex differences in substance use disorder (SUDs) are understudied, and essentially unstudied in terms of the neurobiological underpinnings of sex differences in heroin and cannabis use, as well as in the comorbidity between stress disorders such as PTSD and SUDs. Project 3 serves as the preclinical research arm of the Center. In Aim 1 we will establish the utility of a behavioral model combining an acute life-threatening stress (restraint) in rats with the use and seeking of heroin and cannabis. We will use a novel cannabis addiction model involving self-administration of the two primary cannabinoids found in cannabis, ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD), and along with standard heroin self- administration, examine the acquisition and maintenance of self-administration, as well as context+drug cue induced drug seeking. When rats are pre-exposed to restraint stress or sham 3 weeks prior to beginning self- administration, it will be in combination with an odor that can serve as a stress conditioned stimulus during the drug seeking trial. This model parallels the protocols employed with the cannabis and opioid using human subjects in Center Projects 1 & 2. In Aim 2 of Project 3, the sex differences in drug seeking and interactions with stress will be evaluated for neurobiological mechanisms at the level of plasticity in glutamatergic synapses in the nucleus accumbens core (NAcore). These synapses are known to undergo enduring parallel changes in astroglia and glutamate uptake after drug self-administration that are consequential for cue-induced drug seeking. Moreover, acute stress produces parallel enduring changes that promote the acquisition of drug use. However, sex differences or interactions between stress and sex in this synaptic plasticity are unknown and will be evaluated in Aim 2 using the behavioral model characterized in Aim 1. Finally, in Aim 3 we link directly with the clinical projects by examining drugs to attenuate context+drug cue+stress cue induced heroin and cannabis seeking. Rats will be treated with a compound (N-acetylcysteine) known to ameliorate cue-induced heroin and cannabis seeking by restoring glial glutamate uptake. Separate rats will be administered progesterone or lofexidine in parallel with Center Projects 1 and 2, respectively. Through Aims 1-3 we hope to identify novel mechanisms in synaptic plasticity that will explain interactions between stress, sex and drug seeking. We will also provide mechanistic understanding for how medications used in Projects 1 & 2 successfully reduce stress responsivity in cannabis and opioid use disorders.